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Targeted delivery of ubiquitin-conjugated BH3 peptide-based Mcl-1 inhibitors into cancer cells.

Muppidi A, Doi K, Edwardraja S, Pulavarti SV, Szyperski T, Wang HG, Lin Q - Bioconjug. Chem. (2014)

Bottom Line: J., Gulick, A.Chem.Soc. 134 , 14734 ).

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, State University of New York at Buffalo , Buffalo, New York 14260-3000, United States.

ABSTRACT
BH3 peptides are key mediators of apoptosis and have served as the lead structures for the development of anticancer therapeutics. Previously, we reported the application of a simple cysteine-based side chain cross-linking chemistry to NoxaBH3 peptides that led to the generation of the cross-linked NoxaBH3 peptides with increased cell permeability and higher inhibitory activity against Mcl-1 ( Muppidi, A., Doi, K., Edwardraja, S., Drake, E. J., Gulick, A. M., Wang, H.-G., Lin, Q. ( 2012 ) J. Am. Chem. Soc. 134 , 14734 ). To deliver cross-linked NoxaBH3 peptides selectively into cancer cells for enhanced efficacy and reduced systemic toxicity, here we report the conjugation of the NoxaBH3 peptides with the extracellular ubiquitin, a recently identified endogenous ligand for CXCR4, a chemokine receptor overexpressed in cancer cells. The resulting ubiquitin-NoxaBH3 peptide conjugates showed increased inhibitory activity against Mcl-1 and selective killing of the CXCR4-expressing cancer cells. The successful delivery of the NoxaBH3 peptides by ubiquitin into cancer cells suggests that the ubiquitin/CXCR4 axis may serve as a general route for the targeted delivery of anticancer agents.

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Uptake of ubiquitin–NoxaBH3peptide conjugates is temperature- and CXCR4-dependent. (A) Westernblot analysis of the uptake of PU-KKmt and PU-KKmt-Bpy into Jurkatcells at 37 or 4 °C. The relative uptakes were normalized overβ-actin signal and plotted in the histogram. (B) Western blotanalysis of the uptake of PU-KKmt-Bpy (0.5 μM) into Jurkat cellsat 37 °C after pretreating cells with the CXCR4 antibody (25μg/mL) or ubiquitin at concentrations of 12.5, 25, and 50 μM.The intensities were quantified by densitometry, normalized over β-actinlevels, and plotted in the histogram. (C) PU-KKmt-Bpy uptake at 37°C is dependent on CXCR4 expression. K562, Jurkat, and U937 cellswere incubated with 0.5 μM PU-KKmt-Bpy for 2 h before celllysis and Western blot analysis.
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fig4: Uptake of ubiquitin–NoxaBH3peptide conjugates is temperature- and CXCR4-dependent. (A) Westernblot analysis of the uptake of PU-KKmt and PU-KKmt-Bpy into Jurkatcells at 37 or 4 °C. The relative uptakes were normalized overβ-actin signal and plotted in the histogram. (B) Western blotanalysis of the uptake of PU-KKmt-Bpy (0.5 μM) into Jurkat cellsat 37 °C after pretreating cells with the CXCR4 antibody (25μg/mL) or ubiquitin at concentrations of 12.5, 25, and 50 μM.The intensities were quantified by densitometry, normalized over β-actinlevels, and plotted in the histogram. (C) PU-KKmt-Bpy uptake at 37°C is dependent on CXCR4 expression. K562, Jurkat, and U937 cellswere incubated with 0.5 μM PU-KKmt-Bpy for 2 h before celllysis and Western blot analysis.

Mentions: Since CXCR4-mediated endocytosis is energy-dependentand can be inhibited at low temperatures, we investigated the effectof switching the incubation temperature from 37 to 4 °C on theuptake of PU-KKmt and PU-KKmt-Bpy in the CXCR4-positive Jurkat cells.Because PU-KKmt and PU-KKmt-Bpy contain a His-tag at their N-termini,we measured the internalization of the conjugates by Western blotusing an anti-His tag antibody. We observed >4-fold drop in theuptake for both after lowering the temperature to 4 °C (Figure 4A), indicating that the internalization was energy-dependentand that Bpy-cross-linking did not enhance uptake at either temperature.To confirm that the uptake is mediated through CXCR4, we preincubatedJurkat cells with either anti-CXCR4 antibody or ubiquitin prior toPU-KKmt-Bpy treatment. We found that the pretreatment with anti-CXCR4antibody led to an ∼83% drop in the uptake, while the preincubationwith ubiquitin resulted in a concentration-dependent decrease in theuptake (Figure 4B). These reductions are consistentwith the CXCR4-mediated endocytosis mechanism as both anti-CXCR4 antibodyand ubiquitin can block the ubiquitin binding site of CXCR4 on theJurkat cell surface. The use of a large excess of ubiquitin appearedcritical as the preincubation with 12.5 μM ubiquitin did notinhibit the uptake (Figure 4B). Since the uptakedepends on CXCR4, we evaluated how CXCR4 expression level affectsuptake. We chose three cancer cell lines with the following CXCR4expression order: Jurkat > U937 > K56227 and incubated them with 0.5 μM PU-KKmt-Bpy separately for2 h. The internalization of PU-KKmt-Bpy was quantified by Westernblot using anti-His tag antibody. To our satisfaction, Jurkat cellsshowed the highest uptake, while K562 cells showed the lowest (Figure 4C), indicating that indeed the uptake efficiencycorrelates with the CXCR4 expression level. Taken together, theseresults validated our hypothesis that ubiquitin can serve as a proteincarrier for targeted delivery of anticancer agents such as the cross-linkedpeptides into the CXCR4-expressing cancer cells.


Targeted delivery of ubiquitin-conjugated BH3 peptide-based Mcl-1 inhibitors into cancer cells.

Muppidi A, Doi K, Edwardraja S, Pulavarti SV, Szyperski T, Wang HG, Lin Q - Bioconjug. Chem. (2014)

Uptake of ubiquitin–NoxaBH3peptide conjugates is temperature- and CXCR4-dependent. (A) Westernblot analysis of the uptake of PU-KKmt and PU-KKmt-Bpy into Jurkatcells at 37 or 4 °C. The relative uptakes were normalized overβ-actin signal and plotted in the histogram. (B) Western blotanalysis of the uptake of PU-KKmt-Bpy (0.5 μM) into Jurkat cellsat 37 °C after pretreating cells with the CXCR4 antibody (25μg/mL) or ubiquitin at concentrations of 12.5, 25, and 50 μM.The intensities were quantified by densitometry, normalized over β-actinlevels, and plotted in the histogram. (C) PU-KKmt-Bpy uptake at 37°C is dependent on CXCR4 expression. K562, Jurkat, and U937 cellswere incubated with 0.5 μM PU-KKmt-Bpy for 2 h before celllysis and Western blot analysis.
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Related In: Results  -  Collection

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fig4: Uptake of ubiquitin–NoxaBH3peptide conjugates is temperature- and CXCR4-dependent. (A) Westernblot analysis of the uptake of PU-KKmt and PU-KKmt-Bpy into Jurkatcells at 37 or 4 °C. The relative uptakes were normalized overβ-actin signal and plotted in the histogram. (B) Western blotanalysis of the uptake of PU-KKmt-Bpy (0.5 μM) into Jurkat cellsat 37 °C after pretreating cells with the CXCR4 antibody (25μg/mL) or ubiquitin at concentrations of 12.5, 25, and 50 μM.The intensities were quantified by densitometry, normalized over β-actinlevels, and plotted in the histogram. (C) PU-KKmt-Bpy uptake at 37°C is dependent on CXCR4 expression. K562, Jurkat, and U937 cellswere incubated with 0.5 μM PU-KKmt-Bpy for 2 h before celllysis and Western blot analysis.
Mentions: Since CXCR4-mediated endocytosis is energy-dependentand can be inhibited at low temperatures, we investigated the effectof switching the incubation temperature from 37 to 4 °C on theuptake of PU-KKmt and PU-KKmt-Bpy in the CXCR4-positive Jurkat cells.Because PU-KKmt and PU-KKmt-Bpy contain a His-tag at their N-termini,we measured the internalization of the conjugates by Western blotusing an anti-His tag antibody. We observed >4-fold drop in theuptake for both after lowering the temperature to 4 °C (Figure 4A), indicating that the internalization was energy-dependentand that Bpy-cross-linking did not enhance uptake at either temperature.To confirm that the uptake is mediated through CXCR4, we preincubatedJurkat cells with either anti-CXCR4 antibody or ubiquitin prior toPU-KKmt-Bpy treatment. We found that the pretreatment with anti-CXCR4antibody led to an ∼83% drop in the uptake, while the preincubationwith ubiquitin resulted in a concentration-dependent decrease in theuptake (Figure 4B). These reductions are consistentwith the CXCR4-mediated endocytosis mechanism as both anti-CXCR4 antibodyand ubiquitin can block the ubiquitin binding site of CXCR4 on theJurkat cell surface. The use of a large excess of ubiquitin appearedcritical as the preincubation with 12.5 μM ubiquitin did notinhibit the uptake (Figure 4B). Since the uptakedepends on CXCR4, we evaluated how CXCR4 expression level affectsuptake. We chose three cancer cell lines with the following CXCR4expression order: Jurkat > U937 > K56227 and incubated them with 0.5 μM PU-KKmt-Bpy separately for2 h. The internalization of PU-KKmt-Bpy was quantified by Westernblot using anti-His tag antibody. To our satisfaction, Jurkat cellsshowed the highest uptake, while K562 cells showed the lowest (Figure 4C), indicating that indeed the uptake efficiencycorrelates with the CXCR4 expression level. Taken together, theseresults validated our hypothesis that ubiquitin can serve as a proteincarrier for targeted delivery of anticancer agents such as the cross-linkedpeptides into the CXCR4-expressing cancer cells.

Bottom Line: J., Gulick, A.Chem.Soc. 134 , 14734 ).

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, State University of New York at Buffalo , Buffalo, New York 14260-3000, United States.

ABSTRACT
BH3 peptides are key mediators of apoptosis and have served as the lead structures for the development of anticancer therapeutics. Previously, we reported the application of a simple cysteine-based side chain cross-linking chemistry to NoxaBH3 peptides that led to the generation of the cross-linked NoxaBH3 peptides with increased cell permeability and higher inhibitory activity against Mcl-1 ( Muppidi, A., Doi, K., Edwardraja, S., Drake, E. J., Gulick, A. M., Wang, H.-G., Lin, Q. ( 2012 ) J. Am. Chem. Soc. 134 , 14734 ). To deliver cross-linked NoxaBH3 peptides selectively into cancer cells for enhanced efficacy and reduced systemic toxicity, here we report the conjugation of the NoxaBH3 peptides with the extracellular ubiquitin, a recently identified endogenous ligand for CXCR4, a chemokine receptor overexpressed in cancer cells. The resulting ubiquitin-NoxaBH3 peptide conjugates showed increased inhibitory activity against Mcl-1 and selective killing of the CXCR4-expressing cancer cells. The successful delivery of the NoxaBH3 peptides by ubiquitin into cancer cells suggests that the ubiquitin/CXCR4 axis may serve as a general route for the targeted delivery of anticancer agents.

Show MeSH
Related in: MedlinePlus